Platinum complexes with one radiosensitizing ligand

ABSTRACT

Methods to inhibit tumor growth and to radiosensitize hypoxic cells and pharmaceutical compositions therefor are disclosed. These methods and compositions use compounds of the formula 
     
         PtX.sub.n (NR.sub.2 H) (L)                                 (1) 
    
     wherein n is 1 or 2, and 
     wherein when n is 2, X is a monovalent biologically acceptable anion, and when n is 1, X is a divalent biologically acceptable anion; 
     each R is independently H or alkyl, or both Rs together are a piperidino or morpholino residue; and 
     L is a radiosensitizing ligand selected from a mononitro-substituted imidazole, a mononitro-substituted pyrazole, a mononitro-substituted thiazole and a mononitro-substituted isothiazole.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 037,498,filed Apr. 13, 1987, now U.S. Pat. No. 4,921,963.

TECHNICAL FIELD

The invention relates to the design of metallo-organic complexessuitable for tumor therapy. In particular, it relates to platinumcomplexes containing only one radiosensitizing ligand which areeffective in sensitizing hypoxic cells to radiation, and are also toxicto tumor cells in vivo sensitive to such treatment.

BACKGROUND ART

Apart from surgery, there have been only two general approaches to tumortherapy which have been major viable alternatives in the past twodecades. Although these approaches are far from perfect, sufficientlyacceptable results have been obtained that, despite considerableproblems with side effects and unpredictability of efficacy, thesemethods are, in fact, utilized in the treatment of patients.

These two approaches are, of course, chemotherapy and radiationtreatment. In chemotherapy, the subject is treated with a suitablepharmaceutical substance in protocols which generally do not differmarkedly from administration of other drugs. The tumor selectivity ofthe drug is an inherent function of its chemical nature, and most suchdrugs are chosen because they selectively interfere with rapidly growingcells. The radiation-centered approach can also be aided byadministration of chemical substances to enhance the effectiveness ofthe treatment, i.e., radiosensitizers. This is particularly importantfor treatment of solid tumors using radiation, since the lethal power ofradiation is diminished by the hypoxic nature of most tumors. Hypoxia,in general, renders cell masses relatively resistant to radiation, andthus a mechanism for overcoming this resistance may be supplied in theform of radiosensitizers which, possibly, substitute for oxygen insensitizing the cells to the effects of the radiation.

The number of compounds which have been used as chemotherapeutic agentsis large, and many of the compounds utilized are unrelated to those ofthe present invention. Those most closely related to the inventionherein include the complexes of platinum II of square planarconfiguration, and, in particular, the cis-isomeric forms. As early as1969, cis-platin (cis[PtCl₂ (NH₃)₂ ]) was shown to have antineoplasticactivity. This compound is now widely used in clinics, with secondgeneration compounds in clinical trials since 1979. It has also beenpossible to substitute other metals such as ruthenium and palladium forplatinum as the center ion of the complex, and alternative ligands tothe chloride and amino groups, such as DMSO, ethylenediamine, and soforth, have been studied (Farrell, N. P., et al, Biochem Pharmacol(1984) 33:961-973). The conventional wisdom as stated in the Farrellpaper and elsewhere (Rosenberg, B., in "Cisplatin: Current Status andNew Developments" (1980) Prestayko, A. W., et al, eds, Academic Press,pp. 9 et seq.; Rosenberg, B., Interdisciplinary Science Reviews (1978)3:1-29; Roberts, J. J., in Metal Ions in Genetic Information Transfer(1981) Elsevier, pp. 273 et seq.), is that the cis-isomer is requiredfor antitumor activity.

With regard to radiosensitizing compounds, a number ofmono-nitroimidazole and mono-nitrothiazole derivatives have been shownto exhibit this activity. For example, metronidazole (METRO),misonidazole (MISO), and certain proprietary compounds such as SR-2508(etanidazole) have been shown to have beneficial effects in sensitizingcells to radiation treatment in clinical trials (Overgaard, J., et al,in Proc 3rd Int Meeting on Prog Radio Onc, Raven Press, New York(1986)). METRO and MISO have been studied extensively, and MISO iscommonly used as a standard in in vivo and in vitro tests forradiosensitizing activity (Asquith et al, Radiation Res (1974)60:108-118; Hull et al, Brit J Cancer (1978) 37:567-569; Brown et al,Radiation Res (1980) 82:171-190; U.S. Pat. No. 4,371-540).

Because these compounds are toxic in the amounts needed to beadministered in order for the effect to be exhibited, it has beenattempted to utilize metals known to bind to DNA to carry the organicradiosensitizer to the target. Exemplary reports of this approachinclude Farrell, N. P., et al, Radiation Research (1982) 91:378-379;Bales, J. R., et al, Brit J Cancer (1982) 46:701-705; Bales, J. R., etal, J Chem Soc Chem Comm (1983) 432-433; Chibber, R., et al, Int JRadiation Oncol Biol Phys (1984) 1213-1215; Teicher, D. A., et al, ibid(1984) 11:937-941; Skov, K. A., et al, Proc 7th Intl Cong RadiationResearch (Amsterdam); Broerse, J. J., et al, eds., Abstract B6-29,Martinus Nijhoff (1983), The Hague; Farrell, N. P., et al, Inorg ChimActa (1984) 92:61-66; Chan, P. K. L., Int J Radiation Oncol Biol Phys(1986) 12:1059-1062. All of these reports are directed toradiosensitization studies using metal complexes with knownradiosensitizers in complexes which are bivalent for theradiosensitizing material. In addition, the sensitization of hypoxiccells to radiation as effected by complexes of platinum and relatedmetals with no sensitizing ligand (e.g. Douple, E. B., et al, Brit JCancer Suppl III (1978) 37:98-102) is being exploited clinically. Nias,A. H. W., Int J Radiation Biol (1985) 48:297-314, has presented a reviewof the various approaches to this problem.

The Teicher et al article is particularly relevant because it discloses,among several platinum-containing complexes bisubstituted withradiosensitizers, the mono-substituted platinum complex of1,2-diamino-4-nitrobenzene as a bidentate complex. This compound,designated "Plato" by Teicher, showed a high capacity for killing ofhypoxic cells and less toxicity toward oxygenated cells. In addition,British patent application 2,093,451A and 2,122,194A disclose metalcomplexes of nitro-substituted pyrazoles, nitrazoles, imidazoles, andisothiazoles. These compounds are claimed to be useful in the therapy ofcancer and to have antibacterial activity.

The history of cancer treatment using either direct chemotherapy orradiation with sensitization by compounds of appropriate electronaffinity, such as misonidazole, shows that there is a high variabilitybetween individuals and individual tumors with regard to the sensitivityto particular reagents. That is, it seems to be unpredictable in advancewhat protocols, what sensitizing agents, and what chemotherapeuticagents, if any, will work in an individual instance. Some tumors appearto be easily permanently destroyed by radiation, while other areresistant. Certain tumors are responsive to, for example, cytoxan orprednisone, while others are not. A similar situation is expected toexist with regard to the heavy metal complexes containing organicligands which are toxic to tumors or are radiosensitizers. For example,cisplatin has proven useful in the treatment of testicular cancer, butless so in treatment of cancers at other sites.

Therefore, it becomes crucial to have available a large repertoire ofpossibilities so that the probability that a given subject will respondto at least one or two members of the repertoire will be maximized. Thepresent invention makes a contribution to this repertoire by adding aseries of compounds which have been heretofore unavailable, and whichhave been shown to be effective in radiosensitizing hypoxic cells,including tumors, in model systems, as well as to have tumor toxicity invivo even in the absence of radiation.

DISCLOSURE OF THE INVENTION

The invention compounds are square planar complexes of platinum II, andcontain one radiosensitizing ligand and one or two amine or ammineligands. These compounds are superior to the platinum complexes of theprior art, which bear two radiosensitizing ligands, in their ability tobind DNA and to sensitize hypoxic cells to radiation. In addition, theyare directly toxic to tumor cells in vitro and to tumors in animals. Thein vivo toxicity of the invention compounds is enhanced by treatmentwith the vasoactive antihypertensive agent hydralazine.

Therefore, in one aspect, the invention relates to compounds of theformula: ##STR1## wherein

X is a monovalent biologically

and, in formula (1), X can also represent each "half" of a divalentbiologically acceptable anion;

Y⁻ is a physiologically acceptable anion;

each R is independently H or alkyl, or both Rs together are a piperidinoor morpholino residue; and

L is a radiosensitizing ligand which is a mononitrosubstituted aromaticmoiety containing at least one heterocyclic N and/or substituent amine.Typically, the moiety represented by L is selected from amononitro-substituted imidazole, a mononitro-substituted pyrazole, amononitro-substituted thiazole, a mononitro-substituted isothiazole, anda mononitro-substituted quinoline. Other fused ring systems, such asacridine and terpyridine are also included. These heterocycles mayadditionally be substituted by a substituent R¹ which is selected fromalkyl optionally containing an amino substituent, OR³, and N(R³)₂,wherein R³ is H or lower alkyl. In addition, if the heterocycle isquinoline, pyrazole or imidazole, a ring nitrogen may be substituted byR², which is alkyl of 1-8 carbons substituted by one or more --OR³ andwherein one or two methylenes may be replaced by oxygen.

The radiosensitizing ligands are coordinated to the platinum eitherthrough a ring nitrogen, or, if the heterocyclic ring is substitutedwith an amine or aminoalkyl, through either a ring nitrogen or the aminesubstituent of the ring or alkyl chain.

In other aspects, the invention is directed to pharmaceuticalcompositions containing effective amounts of the compounds of formulas 1or 2, and to methods of treating animal subjects including using thecompounds of these formulas Subjects can be treated using the compoundsof the invention or their pharmaceutical compositions directly inchemotherapeutic regimes, optionally including in these regimes avasoactive agent, or the compounds may be administered to sensitizetumor cells to treatment by radiation.

In addition, the complexes of the invention may be labeled and used forimaging.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the concentration dependence of radiosensitization bycertain compounds of the invention.

FIG. 2 shows the direct toxicity of two compounds of the invention tohypoxic cells in comparison to their toxicity to aerated cells.

FIG. 3 shows the binding to DNA of compounds of the invention asassessed by inhibition of BamHI cleavage of plasmid DNA.

FIG. 4 shows the effect of addition of hydralazine on the ability ofmono-MISO to kill tumor cells in animals.

FIG. 5 is a graph showing the toxicity of the invention compound [PtCl₂(NH₃)(6-nitroquinoline)] as compared to 6-nitroquinoline alone.

FIG. 6 is a graph showing the radiosensitizing effect in vivo of theinvention compound mono-MISO.

FIG. 7 is a graph showing radiosensitization of CHO cells by mono-4NIand mono-5NI.

FIG. 8 is a graph showing DNA binding activity of mono-4NI and mono-5NI.

MODES OF CARRYING OUT THE INVENTION Preferred Embodiments

The compounds of the invention are square planar complexes of platinumII which comprise a single radiosensitizing ligand and one or two ammineor amine substituents. Accordingly, the compounds of the invention maybe prepared in either the cis or trans form. The compounds of formula(1) thus include both of the formulas: ##STR2##

The compounds of formula (2) can be represented by ##STR3##

Both the cis and trans forms are useful in the methods of the invention;however, surprisingly, the trans configuration appears to be somewhatmore effective. The compounds of the invention can thus be used asisolated isomers or as mixtures of both forms. Procedures for convertingthe cis to trans form have been published with regard to analogouscomplexes containing two radiosensitizing ligands (Bales, J. R., et al,J. Chem Soc Chem Com (1983) 432-433) and it has been found that thesemethods are applicable to the complexes containing only one such ligand.

The radiosensitizing ligand is a mono-nitroaromatic moiety. It can be asingle 5- or 6-membered ring, or a fused ring system, including pyrrole,aminophenyl, aminonaphthyl, pyridyl, quinoline, or aminothiophene Theremust be present, of course, at least one of a heterocyclic ring nitrogenor an amino substituent to effect coordination binding of the ligand tothe Pt. In the preferred compounds of the invention, this ligand isselected from mononitro-substituted imidazoles, pyrazoles, thiazoles,isothiazoles, and quinolines, and is coordinated to the platinum througha ring nitrogen as in the formulas below, or through an amino grouprepresented by or included in R¹, if present. If said amino group isavailable, complexes wherein coordination is through this group, andcomplexes wherein coordination is through the ring nitrogen, are bothincluded in the scope of the invention.

Thus, in preferred embodiments, the invention is directed to compoundsof formula 1 wherein L is one of the following: ##STR4## wherein R¹ isalkyl optionally containing an amino substituent, OR³, or N(R³)₂,wherein R³ is H or lower alkyl;

R² is alkyl of 1-8 carbons substituted by one or more --OR³ and whereinone or two methylenes may be replaced by oxygen;

each m independently is 0 or 1.

The single nitro group may be present at positions 2, 4 or 5, forthiazole or imidazole, at positions 3, 4, or 5 for isothiazole orpyrazole, or at any of the ring carbons of quinoline, and the R¹substituent, if present, occupies an alternative site. The nitro groupis a prerequisite for activity, as it supplies the electron affinity tothe ligand. The R¹ substituent is not necessary for activity; hence, therelevant m can be O.

As used herein, "lower alkyl" refers to a saturated, straight orbranched chain hydrocarbyl substituent of 1-4 carbon atoms, for example,methyl, ethyl, n-propyl, i-propyl, n-butyl, sec-butyl, i-butyl, andt-butyl.

"Alkyl" refers to straight, branched chain, or cyclic saturatedhydrocarbyl substituents of 1-8 carbons, such as methyl, i-propyl,n-pentyl, i-pentyl, n-octyl, i-octyl, and 3-methylheptyl and cyclohexyl.

Thus, R¹ may be methyl, ethyl, n-heptyl, i-octyl, amino, methylamino,dimethylamino, hydroxy, methoxy, ethoxy, and the like. For embodimentswhich offer the possibility of coordination of L to the metal through aside chain amino, suitable embodiments for R¹ include 2-aminoethyl,4-amino-n-butyl, 3-aminocyclohexyl, 3-amino-n-pentyl, and the like.

With respect to R², substitution by one or more hydroxyl or lower alkoxygroups on the alkyl group represented by R² or replacement of one ormore methylenes by oxygen is required to enhance the solubility of thecompounds. Convenient embodiments of R² include CH₂ CH(OH)CH₂ OCH₃ andCH₂ CH₂ OH; other preferred embodiments include --CH₂ OCH₂ CHOH.

Particularly preferred compounds of the invention are those wherein L isselected from misonidazole(MISO), which is1-(2-nitro-1-imidazolyl)-3-methoxypropanol and metronidazole (METRO),which is 1,2-hydroxyethyl-2-methyl-5-nitroimidazole. Also preferred arecompounds wherein L is etanidazole (E), 2-amino-5-nitrothiazole (ANT),4-nitroimidazole (4NI), 5-nitro-imidazole (5NI), 6-nitroquinoline (6NQ)or 5-nitroquinoline (5NQ). These embodiments of L are convenient becausethey are widely used in the art and are readily available.

Because 4NI and 5NI are tautomeric, when they are not complexed, they donot exist independently. When complexed with Pt, the tautomeric formsare stabilized and can be separated. (Skov, K. et al, Int J Radiat OncolBiol (1988) in press.)

More particularly preferred are compounds wherein both Rs are H, X isCl, and L is one of these embodiments--i.e., [PtCl₂ (NH₃)L] or[PtCl(NH₃)₂ L] as mono-E, mono-MISO, mono-METRO, mono-ANT, mono-4NI,mono-5NI, mono-5NQ or mono-6NQ or their corresponding di(NH₃) forms.

The amine or ammine substituent(s) of the complexes of the invention ispreferably NH₃. Also included, however, within the scope of theinvention are complexes having substituents which are alkyl ordialkylamines, including cyclic alkylamines, such as cyclohexylamine, orembodiments wherein both Rs together form a part of the ring, such aspiperidine or morpholino. The amine substituent or substituents are,however, not radiosensitizers, and sufficiently neutral to avoidinterference in the ability of the complexes to bind DNA. Indeed it maybe that the one or two am(m)ine substituent(s) are a positive factor inDNA binding.

The "biologically acceptable anion" represented by X and Y⁻, can be anyconvenient organic or inorganic anion which is a suitable leaving groupin vivo. This group is believed to affect the pharmacokinetics of thecompounds, and the compound required for binding may be the aquospecies. Suitable monovalent anions are, for example, chloride, bromide,nitrate, pyruvate, and acetate. Suitable bivalent anions, which can bepresent in Formula (1), include bifunctional organic moieties such asmalonate or 2-ethyl malonate, oxalate, phthalate or substitutedphthalates, and 1,1-cyclobutane dicarboxylate. The sulfate ion can alsobe used in conjunction with a neutral ligand such as water.

Representative compounds of the invention include the cis and transforms, and mixtures thereof, of:

PtCl₂ (ammine)(2-amino-5-nitrothiazole)

PtCl₂ (ammine)(4-nitrothiazole)

PtCl₂ (ammine)(5-i-butyl-2-nitrothiazole)

PtCl₂ (ammine)(2-ethoxy-4-nitrothiazole)

PtCl₂ (ammine)(2-ethoxy-5-nitrothiazole)

PtCl₂ (ammine)(4-dimethylamino-2-nitrothiazole)

PtCl₂ (ammine)(2-t-butoxy-5-nitrothiazole)

PtCl₂ (ammine)(5-(4-ethylhexyl)-2-nitrothiazole)

PtCl₂ (ammine)(4-ethylamino-5-nitrothiazole)

PtCl₂ (ammine)(4-(4-amino-n-butyl)-5-nitrothiazole)

PtCl₂ (ammine)(2-(6-amino-n-hexyl)-5-nitrothiazole)

PtCl₂ (dimethylamino)(2-amino-5-nitrothiazole)

PtCl₂ (dimethylamino)(4-nitrothiazole)

PtCl₂ (dimethylamino)(5-i-butyl-2-nitrothiazole)

PtCl₂ (dimethylamino)(2-ethoxy-4-nitrothiazole)

PtCl₂ (dimethylamino)(2-ethoxy-5-nitrothiazole)

PtCl₂ (dimethylamino)(4-dimethylamino-2-nitrothiazole)

PtCl₂ (dimethylamino)(2-t-butoxy-5-nitrothiazole)

PtCl₂ (dimethylamino)(5-(4-ethylhexyl)-2-nitrothiazole)

PtCl₂ (dimethylamino)(4-ethylamino-5-nitrothiazole)

PtCl₂ (dimethylamino)(4-(4-amino-n-butyl)-5-nitrothiazole)

PtCl₂ (dimethylamino)(2-(6-amino-n-hexyl)-5-nitrothiazole)

PtCl₂ (morpholino)(2-amino-5-nitrothiazole)

PtCl₂ (morpholino)(4-nitrothiazole)

PtCl₂ (morpholino)(5-i-butyl-2-nitrothiazole)

PtCl₂ (morpholino)(2-ethoxy-4-nitrothiazole)

PtCl₂ (morpholino)(2-ethoxy-5-nitrothiazole)

PtCl₂ (morpholino)(4-dimethylamino-2-nitrothiazole)

PtCl₂ (morpholino)(2-t-butoxy-5-nitrothiazole)

PtCl₂ (morpholino)(5-(4-ethylhexyl)-2-nitrothiazole)

PtCl₂ (morpholino)(4-ethylamino-5-nitrothiazole)

PtCl₂ (morpholino)(4-(4-amino-n-butyl)-5-nitrothiazole)

PtCl₂ (morpholino)(2-(6-amino-n-hexyl)-5-nitrothiazole)

PtCl₂ (cyclohexylamino)(2-amino-5-nitrothiazole)

PtCl₂ (cyclohexylamino)(4-nitrothiazole)

PtCl₂ (cyclohexylamino)(5-i-butyl-2-nitrothiazole)

PtCl₂ (cyclohexylamino)(2-ethoxy-4-nitrothiazole)

PtCl₂ (cyclohexylamino)(2-ethoxy-5-nitrothiazole)

PtCl₂ (cyclohexylamino)(4-dimethylamino-2-nitrothiazole)

PtCl₂ (cyclohexylamino)(2-t-butoxy-5-nitrothiazole)

PtCl₂ (cyclohexylamino)(5-(4-ethylhexyl)-2-nitrothiazole)

PtCl₂ (cyclohexylamino)(4-ethylamino-5-nitrothiazole)

PtCl₂ (cyclohexylamino)(4-(4-amino-n-butyl)-5-nitrothiazole)

PtCl₂ (cyclohexylamino)(2-(6-amino-n-hexyl)-5-nitrothiazole)

PtCl₂ (ammine)(2-dimethylamino-5-nitroimidazole)

PtCl₂ (ammine)(4-i-propoxy-2-nitroimidazole)

PtCl₂ (ammine)(2-ethyl-4-nitroimidazole)

PtCl₂ (ammine)(5-n-hexyl-2-nitroimidazole)

PtCl₂ (ammine)(4-methyl-5-nitroimidazole)

PtCl₂ (ammine)(4-nitroimidazole)

PtCl₂ (ammine)(2-nitroimidazole)

PtCl₂ (ammine)(5-nitroimidazole)

PtCl₂ (ammine)(5-hydroxy-2-nitroimidazole)

PtCl₂ (ammine)(4-(4-amino-n-butyl)-5-nitroimidazole)

PtCl₂ (ammine)(2-(6-amino-n-hexyl)-5-nitroimidazole)

PtCl₂ (ammine)(6-nitroquinoline)

PtCl₂ (ammine)(5-nitroquinoline)

PtCl₂ (diethylamino)(2-dimethylamino-5-nitroimidazole)

PtCl₂ (diethylamino)(4-i-propoxy-2-nitroimidazole)

PtCl₂ (diethylamino)(2-ethyl-4-nitroimidazole)

PtCl₂ (diethylamino)(5-n-hexyl-2-nitroimidazole)

PtCl₂ (diethylamino)(4-methyl-5-nitroimidazole)

PtCl₂ (diethylamino)(4-nitroimidazole)

PtCl₂ (diethylamino)(2-nitroimidazole)

PtCl₂ (diethylamino)(5-nitroimidazole)

PtCl₂ (diethylamino)(5-hydroxy-2-nitroimidazole)

PtCl₂ (dimethylamino)(4-(4-amino-n-butyl)-5-nitroimidazole)

PtCl₂ (dimethylamino)(2-(6-amino-n-hexyl)-5-nitroimidazole)

PtCl₂ (dimethylamino)(6-nitroquinoline)

PtCl₂ (dimethylamino)(5-nitroquinoline)

PtCl₂ (morpholino)(2-dimethylamino-5-nitroimidazole)

PtCl₂ (morpholino)(4-i-propoxy-2-nitroimidazole)

PtCl₂ (morpholino)(2-ethyl-4-nitroimidazole)

PtCl₂ (morpholino)(5-n-hexyl-2-nitroimidazole)

PtCl₂ (morpholino)(4-methyl-5-nitroimidazole)

PtCl₂ (morpholino)(4-nitroimidazole)

PtCl₂ (morpholino)(2-nitroimidazole)

PtCl₂ (morpholino)(5-nitroimidazole)

PtCl₂ (morpholino)(5-hydroxy-2-nitroimidazole)

PtCl₂ (morpholino)(4-(4-amino-n-butyl)-5-nitroimidazole)

PtCl₂ (morpholino)(2-(6-amino-n-hexyl)-5-nitroimidazole)

PtCl₂ (morpholino)(6-nitroquinoline)

PtCl₂ (morpholino)(5-nitroquinoline)

PtCl₂ (cyclohexylamino)(2-dimethylamino-5-nitroimidazole)

PtCl₂ (cyclohexylamino)(4-i-propoxy-2-nitroimidazole)

PtCl₂ (cyclohexylamino)(2-ethyl-4-nitroimidazole)

PtCl₂ (cyclohexylamino)(5-n-hexyl-2-nitroimidazole)

PtCl₂ (cyclohexylamino)(4-methyl-5-nitroimidazole)

PtCl₂ (cyclohexylamino)(4-nitroimidazole)

PtCl₂ (cyclohexylamino)(2-nitroimidazole)

PtCl₂ (cyclohexylamino)(5-nitroimidazole)

PtCl₂ (cyclohexylamino)(5-hydroxy-2-nitroimidazole)

PtCl₂ (cyclohexylamino)(4-(4-amino-n-butyl)-5-nitroimidazole)

PtCl₂ (cyclohexylamino)(2-(6-amino-n-hexyl)-5-nitroimidazole)

PtCl₂ (cyclohexylamino)(6-nitroquinoline)

PtCl₂ (cyclohexylamino)(5-nitroquinoline)

PtCl₂ (ammine)(3-amino-5-nitroisothiazole)

PtCl₂ (ammine)(4-nitroisothiazole)

PtCl₂ (ammine)(5-i-butyl-3-nitroisothiazole)

PtCl₂ (ammine)(3-ethoxy-4-nitroisothiazole)

PtCl₂ (ammine)(3-ethoxy-5-nitroisothiazole)

PtCl₂ (ammine)(4-dimethylamino-3-nitroisothiazole)

PtCl₂ (ammine)(3-t-butoxy-5-nitroisothiazole)

PtCl₂ (ammine)(5-(4-ethylhexyl)-3-nitroisothiazole)

PtCl₂ (ammine)(4-ethylamino-5-nitroisothiazole)

PtCl₂ (ammine)(4-(4-amino-n-butyl)-5-nitroisothiazole)

PtCl₂ (ammine)(3-(6-amino-n-hexyl)-5-nitroisothiazole)

PtCl₂ (dimethylamino)(3-amino-5-nitroisothiazole)

PtCl₂ (dimethylamino)(4-nitroisothiazole)

PtCl₂ (dimethylamino)(5-i-butyl-3-nitroisothiazole)

PtCl₂ (dimethylamino)(3-ethoxy-4-nitroisothiazole)

PtCl₂ (dimethylamino)(3-ethoxy-5-nitroisothiazole)

PtCl₂ (dimethylamino)(4-dimethylamino-3-nitroisothiazole)

PtCl₂ (dimethylamino)(3-t-butoxy-5-nitroisothiazole)

PtCl₂ (dimethylamino)(5-(4-ethylhexyl)-3-nitroisothiazole)

PtCl₂ (dimethylamino)(4-ethylamino-5-nitroisothiazole)

PtCl₂ (dimethylamino)(4-(4-amino-n-butyl)-5-nitroisothiazole)

PtCl₂ (dimethylamino)(3-(6-amino-n-hexyl)-5-nitroisothiazole)

PtCl₂ (morpholino)(3-amino-5-nitroisothiazole)

PtCl₂ (morpholino)(4-nitroisothiazole)

PtCl₂ (morpholino)(5-i-butyl-3-nitroisothiazole)

PtCl₂ (morpholino)(3-ethoxy-4-nitroisothiazole)

PtCl₂ (morpholino)(3-ethoxy-5-nitroisothiazole)

PtCl₂ (morpholino)(4-dimethylamino-3-nitroisothiazole)

PtCl₂ (morpholino)(3-t-butoxy-5-nitroisothiazole)

PtCl₂ (morpholino)(5-(4-ethylhexyl)-3-nitroisothiazole)

PtCl₂ (morpholino)(4-ethylamino-5-nitroisothiazole)

PtCl₂ (morpholino)(4-(4-amino-n-butyl)-5-nitroisothiazole)

PtCl₂ (morpholino)(3-(6-amino-n-hexyl)-5-nitroisothiazole)

PtCl₂ (cyclohexylamino)(3-amino-5-nitroisothiazole)

PtCl₂ (cyclohexylamino)(4-nitroisothiazole)

PtCl₂ (cyclohexylamino)(5-i-butyl-3-nitroisothiazole)

PtCl₂ (cyclohexylamino)(3-ethoxy-4-nitroisothiazole)

PtCl₂ (cyclohexylamino)(3-ethoxy-5-nitroisothiazole)

PtCl₂ (cyclohexylamino)(4-dimethylamino-3-nitroisothiazole)

PtCl₂ (cyclohexylamino)(3-t-butoxy-5-nitroisothiazole)

PtCl₂ (cyclohexylamino)(5-(4-ethylhexyl)-3-nitroisothiazole)

PtCl₂ (cyclohexylamino)(4-ethylamino-5-nitroisothiazole)

PtCl₂ (cyclohexylamino)(4-(4-amino-n-butyl)-5-nitroisothiazole)

PtCl₂ (cyclohexylamino)(3-(6-amino-n-hexyl)-5-nitroisothiazole)

PtCl₂ (ammine)(3-dimethylamino-5-nitropyrazole)

PtCl₂ (ammine)(4-i-propoxy-3-nitropyrazole)

PtCl₂ (ammine)(3-ethyl-4-nitropyrazole)

PtCl₂ (ammine)(5-n-hexyl-3-nitropyrazole)

PtCl₂ (ammine)(4-methyl-5-nitropyrazole)

PtCl₂ (ammine)(4-nitropyrazole)

PtCl₂ (ammine)(3-nitropyrazole)

PtCl₂ (ammine)(5-nitropyrazole)

PtCl₂ (ammine)(etanidazole)

PtCl₂ (ammine)(5-hydroxy-3-nitropyrazole)

PtCl₂ (ammine)(4-(4-amino-n-butyl)-5-nitropyrazole)

PtCl₂ (ammine)(3-(6-amino-n-hexyl)-5-nitropyrazole)

PtCl₂ (diethylamino)(3-dimethylamino-5-nitropyrazole)

PtCl₂ (diethylamino)(4-i-propoxy-3-nitropyrazole)

PtCl₂ (diethylamino)(3-ethyl-4-nitropyrazole)

PtCl₂ (diethylamino)(5-n-hexyl-3-nitropyrazole)

PtCl₂ (diethylamino)(4-methyl-5-nitropyrazole)

PtCl₂ (diethylamino)(4-nitropyrazole)

PtCl₂ (diethylamino)(3-nitropyrazole)

PtCl₂ (diethylamino)(5-nitropyrazole)

PtCl₂ (diethylamino)(etanidazole)

PtCl₂ (diethylamino)(5-hydroxy-3-nitropyrazole)

PtCl₂ (dimethylamino)(4-(4-amino-n-butyl)-5-nitropyrazole)

PtCl₂ (dimethylamino)(3-(6-amino-n-hexyl)-5-nitropyrazole)

PtCl₂ (morpholino)(3-dimethylamino-5-nitropyrazole)

PtCl₂ (morpholino)(4-i-propoxy-3-nitropyrazole)

PtCl₂ (morpholino)(3-ethyl-4-nitropyrazole)

PtCl₂ (morpholino)(5-n-hexyl-3-nitropyrazole)

PtCl₂ (morpholino)(4-methyl-5-nitropyrazole)

PtCl₂ (morpholino)(4-nitropyrazole)

PtCl₂ (morpholino)(3-nitropyrazole)

PtCl₂ (morpholino)(5-nitropyrazole)

PtCl₂ (morpholino)(etanidazole)

PtCl₂ (morpholino)(5-hydroxy-3-nitropyrazole)

PtCl₂ (morpholino)(4-(4-amino-n-butyl)-5-nitropyrazole)

PtCl₂ (morpholino)(3-(6-amino-n-hexyl)-5-nitropyrazole)

PtCl₂ (cyclohexylamino)(3-dimethylamino-5-nitropyrazole)

PtCl₂ (cyclohexylamino)(4-i-propoxy-3-nitropyrazole)

PtCl₂ (cyclohexylamino)(3-ethyl-4-nitropyrazole)

PtCl₂ (cyclohexylamino)(5-n-hexyl-3-nitropyrazole)

PtCl₂ (cyclohexylamino)(4-methyl-5-nitropyrazole)

PtCl₂ (cyclohexylamino)(4-nitropyrazole)

PtCl₂ (cyclohexylamino)(3-nitropyrazole)

PtCl₂ (cyclohexylamino)(5-nitropyrazole)

PtCl₂ (cyclohexylamino)(etanidazole)

PtCl₂ (cyclohexylamino)(5-hydroxy-3-nitropyrazole)

PtCl₂ (cyclohexylamino)(4-(4-amino-n-butyl)-5-nitropyrazole)

PtCl₂ (cyclohexylamino)(3-(6-amino-n-hexyl)-5-nitropyrazole)

and the corresponding bromides, nitrates, pyruvates, acetates,malonates, ethyl malonates, oxalates, phthalates, and 1,1-cyclobutanedicarboxylates.

To the foregoing list, which includes the compounds of Formula (1), isadded the corresponding list of compounds of Formula (2) wherein eachentry has the formula

    PtCl(ammine).sub.2 L.sup.+ Y.sup.-

    PtCl(dimethylamino).sub.2 L.sup.+ Y.sup.-

    PtCl(morpholino).sub.2 L.sup.+ Y.sup.-

    PtCl(cyclohexylamino).sub.2 L.sup.+ Y.sup.-

in place of the moieties shown. For these compounds of Formula (2), eachNHR₂ ligand is independent; also included are, e.g.,PtCl(ammine)(morpholino)L, PtCl(morpholino)(cyclohexylamino)L, etc.Nitrate is preferred for Y⁻.

SYNTHESIS OF THE INVENTION COMPOUNDS

The compounds of formula (1) are synthesized from the monoammine ofPt(II) of the formula K[PtCl₃ (NH₃)] by reaction of one equivalent ofthe radiosensitizing ligand in aqueous solution, according to thescheme:

    K[PtCl.sub.3 (NH.sub.3)]+L $ Pt(L)(NH.sub.3)Cl.sub.2 +KCl

The reaction is allowed to proceed for 10-36, preferably 12-24, hours,and the complex formed may be recovered by evaporation of the solventand crystallization. The resulting compounds of Formula (1) aremono-substituted with L, and are predominantly in the cis form. Thetrans form is obtained by redissolving in a suitable solvent, such asethanol, and permitting isomerization to take place.

The compounds of Formula (2) are prepared by treatment of the dichlorodiamine with the radiosensitizing ligand in the presence of silvernitrate, according to the reaction

    [PtCl.sub.2 (NH.sub.3).sub.2 ]+L+Ag.sup.+ $[PtCl(NH.sub.3).sub.2 L].sup.+ +AgCl.

The AgCl is removed by filtration and the complex recovered byevaporation of solvent and crystallization.

FORMULATION AND ADMINISTRATION

The platinum complexes of Formulas (1) and (2) mono-substituted withradiosensitizers are useful either for direct chemotherapy or tosensitize hypoxic tumor cells in warmblooded animal hosts to radiationor to other drugs. While humans are the subject of most concern, thecompounds of the invention are also useful in treatment of animalsubjects such as farm animals, sports animals, and pets.

For use as radiosensitizers, the compounds are administered to subjectswither systemically or locally. Systemic administration may be oral orparenteral, and typically intravenous administration is preferred. Forparenteral administration, the compounds of Formulas (1) or (2) aregenerally prepared in a unit dosage-injectable form as a solution,emulsion, suspension, as so forth with a pharmaceutically acceptableexcipient. Excipients and formulations are known in the art and aresummarized, for example, in Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., latest edition. Examples of excipientsinclude water, buffer, saline, Ringer's solution, dextrose, and Hanks'solution. Nonaqueous vehicles may also be included, such as peanut orcottonseed oil, or single compounds such a triglycerides or fatty acidesters. Additional substances such as stabilizers, buffers,antioxidants, and preservatives may also be included in the composition.

Oral administration may be by capsule or pill or powder, and may containadditional diluents such as lactose, maltose, starch, gelatin, sorbitan,and magnesium stearate.

The amount of compound administered is sufficient to sensitize thetarget cells, but less than an amount toxic to the organism. The amountwill depend, of course, on the individual subject, the tumor beingtreated, the protocol for radiation, and the judgment of thepractitioner. In-most radiation protocols, a fractionated radiationdosage is given over a period of several days, weeks, or months, and thecompounds of the invention are administered in conjunction with theradiation. They are administered at a time such that their peakconcentration in the hypoxic cells occurs at the time radiationtreatment is administered. Clearly, the timing of this administration issubject to considerable variation, depending, for example, on the dosageform and the location of the tumor. Individual protocols should bedesigned for particular situations and individual patients, but suchdesign is well within the skill of the practitioner of the art, andrequires little in the way of calibration and verification.

In view of the foregoing, it is difficult to estimate a generalizeddosage, but most protocols will be effective with dosages in the rangeof 0.01-20 mmol of the radiosensitizing compounds per kg of subject'sweight.

Exemplary carcinomas and sarcomas which are susceptible toradiosensitization include carcinomas of the intestines, liver, lung,skin, and cervix, and sarcomas such as Ewing's, lymphatic, Jensen's,Karposi's, and the like.

For use in chemotherapy, the compounds are administered in accordancewith usual procedures for drug administration and formulation, such asthose set forth in Remington's Pharmaceutical Sciences, referencedabove.

Chemotherapeutic protocols are aided by the introduction into theprotocol of a suitable vasoactive agent, such as 5-hydroxytrypamine(5-HT) or hydralazine, preferably hydralazine. It has been shown, forexample, that the amount of RSU-1069 required for effectiveness can bereduced by administration of 5-hydroxytryptamine or hydralazine(Chaplin, D. J., et al, Brit J Cancer (1986) 54:727-731; Horwich, A., etal, Brit J Radiol (1986) 59:1238-1240). RSU-1069 is a compound sometimesused for chemotherapy and having a high incidence of side effects. Thereagents noted are effective in reducing blood flow to tumors withoutaffecting normal tissue blood flow. Other vasoactive agents could alsobe used.

The compounds of the invention also show enhanced activity incytotoxicity to hypoxic cells in the presence of vasoactive agents. Theadministration of such agents can further reduce the oxygenation oftumors, and thereby intensify the cyctotoxic effects of agents which areselective for hypoxic cells. Accordingly, in a preferred method, thecompounds of the invention are administered to the subject at reduceddosage levels, along with an effective amount of 5-HT or hydralazine, orof other suitable vasoactive agents which selectively reduce blood flowto tumors.

Typical dosages of the compounds of the invention for chemotherapeuticuse without the administration of the vasoactive agent are in the rangeof approximately 0.1-5 mg/kg. Reductions in dosage levels ofapproximately ten-fold may be achieved by coadministration ofappropriate vasoactive agents. Conversely, the efficacy of the inventivecompounds can be improved by coadministration with vasoactive agents atthe same dosage levels.

Tumors susceptible to chemotherapeutic treatment with the compounds ofthe invention in either the presence or absence of vasoactive agents arehypoxic solid tumors, and include a list of specific tumor types similarto that set forth above for the radiosensitizing protocols.

In addition, the compounds of the invention are useful for radioimagingof hypoxic tumors. The compounds are combined with a scintographiclabel, such as ⁹⁹ Tc, ¹³¹ I, or ¹¹¹ n using standard labelingtechniques, and the labeled complexes administered systemically. Thelabeled complexes home to the location of the hypoxic cells and can bedetected using conventional scanning techniques such as those describedin Franco, H., et al, Int J Radiat Onco Biol Phys (1986) 12:1195-1202.

EXAMPLES

The following examples are intended to illustrate but not to limit theinvention.

EXAMPLE 1 Preparation of Cis and Trans Mono-MISO

One equivalent of misonidazole (205 mg, 1 mM) was added to a solution ofK[PtCl₃ (NH₃)], (358.35 mg, 1 mM), dissolved in the minimum volume of H₂O using an equal volume of hot H₂ O (The starting material K[PtCl₃(NH₃)] was prepared from K₂ PtCl₄ according to the method of Abrams, M.et al, Inorganic Chemistry (in press), but may be prepared by otherknown methods.)

The mixture was then heated until all the nitroimidazole ligand wasdissolved, during which time the solution color changed from yellow toorange. The solution was then stirred for approximately 24 hr at roomtemperature and evaporated to dryness. The resultant orange oil wasdissolved in acetone and the solution filtered to remove KCl.

The filtrate was dried over anhydrous CaCl₂, filtered again, andevaporated to dryness. The residue was treated with diethyl ether togive the yellow-orange product PtCl₂ (NH₃) (misonidazole) (mono-MISO),yield 60%, which was designated as cis based on IR spectral dataElemental analysis: %C, 17.40 (17.35); %H, 3.02 (2.89); %N, 11.39(11.57).

To obtain the trans configuration, the orange solid was dissolved inEtOH by gentle warming. Upon dissolution, the initial orange colorchanged to yellow-green and, upon cooling, a yellow product, which wasthe trans configuration based on IR spectral data, which precipitatesout at 50% yield. Elemental analysis: %C, 17.32 (17.35); %H, 2.93(2.89); %N, 10.99 (11.57); %Cl, 15.57 (14.67). MW =488 (calc: 484).

The IR spectrum for the cis configuration showed a broad band withmaxima at 338 and 332 cm⁻¹. The trans configuration showed a sharperband at 330 cm⁻¹, different from the cis configuration. Otherdifferences were also observed. The trans compound shows extra bands at2400-3500 cm⁻¹.

EXAMPLE 2 Preparation of Mono-METRO

In a manner exactly analogous to that set forth in Example 1, butsubstituting for misonidazole, metronidazole, the cis form of mono-METROwas prepared in 55% yield. Elemental analysis: %C, 15.76 (15.86); %H,2.31 (2.64); %N, 12.06 (12.33); %Cl, 15.95 (15.62). Molecular weight inmethanol, 429 (calc. 453). A broad band for (PtCl) with maxima at 339and 331 cm⁻¹ was obtained in the IR spectrum.

In a manner similar to that set forth in Example 1, the cis-isomer wasdissolved in ethanol and warmed to convert at least a portion of thematerial to the trans form, which was recovered as described in Example1.

EXAMPLE 3 Preparation of Mono-NI

In a manner similar to that set forth in Example 1, but substituting4-nitroimidazole for misonidazole, the cis-platinum complex coordinatedwith one ligand of 4-nitroimidazole (mono-NI) was obtained, andcharacterized by elemental analysis as: %C, 8.33 (9.09); %H, 1.32 (1.5);%N, 10.84 (10.6).

EXAMPLE 4 Preparation of Mono-ANT

In a manner precisely similar to that set forth in Example 1, butsubstituting for misonidazole 2-amino-5-nitrothiazole, the correspondingcis and trans forms of mono-ANT were prepared.

EXAMPLE 5 Comparison of Toxicity in Hypoxic and Aerated Cells

The toxicity of the invention compounds with respect to Chinese hamsterovary (CHO) cells was compared in air and in nitrogen induced hypoxia asdescribed by Moore, B. A., et al, Radiation Research (1976) 67:459-478.The radiosensitizing ligands, or cis or transdichlorodiamine platinum II(DDP) or the . bisubstituted platinum complexes, PtCl₂ (L)₂, were usedas controls.

The cells were carried in MEM a medium with 10% fetal calf serum, andplating efficiencies were determined on the seventh day after plating.

In one set of experiments, the toxicity of the cis and trans-mono-MISOcomplexes was determined at 100 mM/dm³ in aerobic and hypoxic cells.These results are shown in FIG. 1. It is clear from FIG. 1 that toxicityis greater in hypoxia than in air, and that the trans isomer is moretoxic than cis. Either complex is more toxic than MISO alone, althoughless toxic than cisDDP, as indicated by the dashed line for cis-DDP at10 mM/dm³.

In similar experiments at 100 mM/dm³, MISO and trans-[PtCl₂ (miso)₂ ]show no toxicity. Survival rates for trans-DDP and monoammine were 0.2and 0.1 respectively. Thus, cis-DDP and the monoammine were somewhatmore toxic than the compounds of the invention; trans-[PtCl₂ (miso)₂ ]and trans-DDP are somewhat less toxic. The approximate order of hypoxictoxicity of platinum complexes is cis-DDP ff trans-mono-MISO fmonoammine Z cis-mono-MISO f trans-DDP f mono-METRO Z mono-NI fmono-ANT. The order of toxicity of free ligand is 4-nitroimidazole fMISO f ANT f METRO.

In similar experiments using the corresponding 6-nitroquinolinecompound, [PtCl₂ (NH₃)(6NQ)], (mono-6NQ), the complex showed much highertoxicity than 6NQ alone, as demonstrated in FIG. 5. Using 100 mM levels,mono-6NQ caused plating efficiency to drop to 0 after an hour, while 6NQhad no effect after 3 hours.

EXAMPLE 6 Radiosensitization of CHO Cells

Radiosensitization of CHO cells was determined as described by Moore, B.A., et al, sucra, at 4° C., except that the cells were incubated withcomplex for 1 hr at 37° C. to allow binding to DNA prior to radiation. APicker X-ray source (250 kVp, 1.7 mm Cu) was used. As with the toxicitystudies, cells were carried, irradiated, and plated in MEM a medium with10% fetal calf serum, and plating efficiencies were determined on theseventh day after plating. Enhancement ratios (ER) were calculated fromthe doses required to reduce survival to 1%.

FIG. 2 shows the results obtained for three compounds of the invention,mono-MISO, mono-NI, and mono-ANT, all in the cis configuration atvarious concentrations. Mono-MISO was somewhat more effective thanmono-NI, which was somewhat more effective than mono-ANT. Mono-METRO,not shown in the graph, was approximately equal to mono-ANT. At 100mM/dm³, the alone); mono-NI gave 1.16 (vs. 1.10 for ligand alone);mono-ANT gave 1.15 (vs. 1.25 for ligand alone). Sensitization was alsotested in oxygen for the various compounds, and no sensitization wasobtained by trans-bis-MISO, MISO alone, or cis-mono-MISO.

Table 1 summarizes the results at various concentrations for thecompounds tested.

                  TABLE 1                                                         ______________________________________                                                        mmol/dm.sup.3                                                                         ER                                                    ______________________________________                                        Monoammine        100       1.2                                               cis-mono-MISO     100        1.25                                             trans-mono-MISO   100        1.25                                             trans-bis-MISO    100       1.2                                               trans-DDP         100       1.3                                               cis-DDP            10       1.3                                               MISO              100       1.3                                                                 250       1.4                                               ______________________________________                                    

The ability of cis-mono-4NI and of cis-mono-5NI to radiosensitize CHOcells in comparison with free 4(5)-nitroimidazole is shown in FIG. 7.The enhancement ratio (ER) was higher for mono-5NI than for mono-4NI(1.28 as compared to 1.16).

Radiosensitization by mono-MISO was also shown in vivo in the KHT tumormodel using 200 mg/kg of trans mono-MISO administered 3, 2, 1, 1/2 or 0hours prior to X-irradiation. The results shown in FIG. 6, indicate drugadministered 0.5 or 1 hour prior to irradiation enhance the effect ofthe X-rays.

EXAMPLE 7 Binding to DNA

The ability of the invention compounds to bind DNA was tested using anassay which is based on inhibition of the ability of restriction enzymesto cleave the complexed DNA. The substrate in the assay was the plasmidpSV2-gpt (Mulligan, R., et al Science (1980) 209:1422-1427). Aftergrowth in E. coli. this 5.2 kb plasmid was extracted and linearizedusing PvuII. Cleavage of the extracted linearized segment by BamHI orEcoRI results in two fragments per enzyme. Inhibition of this cleavageby test compounds provides an assay method for binding of the testcompounds.

In the assay, DNA (20 mg/ml) in 100 mM Tris-HCl, 1 mM EDTA, pH 8, wastreated with the test compounds at approximately 100 mM for 1 hr at 37°C. Unbound drug was then removed using a G-50 Sephadex spin column(Pharmacia).

To 30 mM of the treated DNA, 23.5 ml of a buffer solution containing0.33 M Tris-acetate, pH 7.9, 0.66 M potassium acetate, 0.10 Mdithiothreitol, and 1 mg/ml BSA, along with 10 units of enzyme, wereadded. Enzyme activity was stopped using 1 ml 0.5 M EDTA after 30 min at37° C. Tracking 0.25% bromophenol blue, 0.25% xylene cyanol in 30%glycerol water) was added, and 10 ml of the resulting solution loadedonto a 1% agarose gel made with E buffer (0.04 M Tris, 5 mM sodiumacetate; 1 mM EDTA, pH 7.8) and subjected to horizontal electrophoresis.The slab gel was stained with aqueous ethidium bromide solution (1ng/ml) and photographed under UV light.

Inhibition of the restriction enzyme cleavage was assessed qualitativelyby noting the relative proportion of bands, or quantitatively byanalyzing densitometric scans of the negatives of the recorded pictures.

The controls, cis-DDP and monoammine, showed binding to the DNA asindicated by enzyme inhibition; cis-DDP binds much more strongly thanthe monoammine. Thus, treatment with only 40 mM cis-DDP results in a 30%inhibition of BamHI cleavage, a level not reached by treatment of theDNA with the monoammine until the concentration was increased to 160 mM.The results for the compounds of the invention are shown in FIG. 3. Atleast 10% inhibition of cleavage with BamHI was obtained for mono-ANT at320 mM, but for mono-METRO and mono-MISO this is attained at a muchlower concentration of approximately 50 mM.

In a similar determination, the results of which are shown in FIG. 8,cis-mono-5NI appeared to show more effective binding to DNA at allconcentrations than cis-mono-4NI; neither bound as well, however, ascis-DDP.

These results are consistent with data obtained in whole cells usingatomic absorption. In this procedure, after exposure to the testcompound for 1-2 hr, 10⁹ CHO cells previous for 18 hr with ¹⁴ Cthymidine were washed and their DNA extracted, according to theprocedure of Pera, M. F., Jr., et al, Chem Biol Interact (1981)37:245-261, and analyzed for platinum using a Perkin-Elmer AtomicAbsorption Spectrometer with a graphite furnace. ¹⁴ C counts were usedto normalize the platinum results by counting an aliquot of thehydrolyzate which had been prepared for atomic absorption in liquidscintillation counter and comparing these ¹⁴ C counts to the number ofcounts per cell. For treatment with the various compounds of theinvention at 100 mM, the amount of platinum bound to the extracted DNAwas, in ng Pt/10⁶ cells: 6 for cis-DDP, 5 for trans-DDP, andconsiderably higher for some of the sensitizer containing platinumcomplexes. Though the results of this assay were erratic they establishthat the Pt complexes cross cell membranes and bind to DNA in mammaliancells.

EXAMPLE 8 Potentiation of Cytotoxicity Using Vasodilators

The effect of the vasodilator hydralazine on the cyctoxicity of thecompounds of the invention for toxic cells in vivo was tested using theLewis lung carcinoma or KHT sarcoma model in C57B1 mice. The protocolswere those of Chaplin, D. J. (Brit J Cancer (1986) 54:727-731).

When these procedures were applied to potentiation by hydralazine ofRSU-1069 (Chaplin, D. J., et al, Int J Radiat Oncol Biol Phys (1987), inpress), hydralazine was shown, at levels of 5 mg/kg or 30 mg/kg, toenhance the effect of RSU-1069 administered at 0.1 mg/g IP ifadministered within three hours of the administration of the RSU-1069.Similar protocols using the invention compound mono-MISO showedpotentiating effects of hydralazine on this compound at 5 mg/kg, asshown in FIG. 4. These results pertain to the KHT sarcoma model;mono-MISO was administered at doses between 10 and 300 mg/kg with orwithout supplement of hydralazine. As indicated in FIG. 4, the cellsurvival drops drastically (approx. 3 logs) in the presence of thecombined therapy. A much lower dose (4×) of the invention compounds areneeded to achieve equitoxic doses when hydralazine is coadministered.

EXAMPLE 9 Preparation of [PtCl₂ (NH₃)(6-nitroquinoline)

A. 6-nitroquinoline (0.26 g) was dissolved in 30 ml methanol and addedto a solution containing 0.487 g K[PtCl₃ NH₃ ] in 30 ml of water. Thesolution was stirred overnight and the next day the yellow precipitatewas filtered, washed with water ethanol and ether, and recrystalizedfrom DMF/water.

Elemental analysis: C: 23.6 (calculated: 23.6); H: 1.9 (calculated1.98); N: 9.1 (calculated: 9.2)

B. PtCl₂ (NH₃) (5-nitroquinoline) was prepared in an identical manner,substituting 5-nitroquinoline for 6-nitroquinoline.

EXAMPLE 10 Preparation of [PtCl(NH₃)₂ (metronidazole)]⁺ NO₃ ⁻

To a suspension of cis-[PtCl₂ (NH₃)₂ ] in water was added 1 equivalentof metronidazole, also in water, followed by 1 equivalent of AgNO₃ inmethanol (water to methanol final volume 1:1). The reaction was stirredovernight. The colorless solution was then filtered to remove AgCl andthe solution was evaporated to 1/2 volume. The product title compoundwas precipitated by addition of acetone.

EXAMPLE 11 Cross-Resistance with Other Drugs

Patients become resistant to cisplatin (cisDDP) and to many relateddrugs. Several of the invention compounds were tested for toxicity withrespect to cisDDP-sensitive and cisDDP-resistant cells (according to themethod of Hacker, et al, Cancer Res (1985) 45:4748.) As shown in Table2, these compounds are comparably effective in both, while cisDDP is 50×more effective in "sensitive" cells. The data in Table 2 thus show thatthe "mono" series do not show cross-resistance.

                  TABLE 2                                                         ______________________________________                                        ID.sub.50 in L1210 Cells                                                                  cDDP sensitive                                                                             cDDP resistant                                       Complex     line mM      line mM     Ratio                                    ______________________________________                                        cis mono MISO                                                                             8.5          f41         5                                        trans mono MISO                                                                           6.5          18.6        3                                        cis mono METRO                                                                            4.4          f23.12      5                                        cisDDP      0.1          5           50                                       transDDP    22           66          3                                        ______________________________________                                    

We claim:
 1. A pharmaceutical composition for sensitization of hypoxiccells to radiation or for the inhibition of tumor growth in those tumorssensitive to treatment therewith which comprises an effective amount ofthe compound of the formula:

    PtX.sub.n (NR.sub.2 H)(L)                                  (1)

wherein n is 1 or 2, and wherein when n is 2, X is a monovalentbiologically acceptable anion, and when n is 1, X is a divalentbiologically acceptable anion; each R is independently H or alkyl, orboth Rs together are a piperidino or morpholino residue; and L is aradiosensitizing ligand selected from a mononitro-substituted imidazole,a mononitro-substituted pyrazole, a mononitro-substituted thiazole and amononitro-substituted isothiazole, in admixture with a suitablepharmaceutically acceptable excipient.
 2. A method to inhibit tumorgrowth in those tumors sensitive to treatment therewith in animals whichcomprises administering to an animal in need of such treatment atumor-inhibiting amount of a compound of the formula:

    PtX.sub.n (NR.sub.2 H)(L)                                  (1)

wherein n is 1 or 2, and wherein when n is 2, X is a monovalentbiologically acceptable anion, and when n is 1, X is a divalentbiologically acceptable anion; each R is independently H or alkyl, orboth Rs together are a piperidino or morpholino residue; and L is aradiosensitizing ligand selected from a mononitro-substituted imidazole,a mononitro-substituted pyrazole, a mononitro-substituted thiazole and amononitro-substituted isothiazole or a pharmaceutical compositioncontaining a tumor-inhibiting amount of the compound of formula
 1. 3.The method of claim 2 which further includes the administration of avasoactive agent to said animal.
 4. The method of claim 3 wherein thevasoactive agent is hydralazine.
 5. A method to inhibit the growth ofhypoxic tumors in those tumors sensitive to treatment therewith in awarmblooded animal which method comprises radiosensitizing the hypoxictumor cells in said animal by administering the compound of the formula:

    PtX.sub.n (NR.sub.2 H)(L)                                  (1)

wherein n is 1 or 2, and wherein when n is 2, X is a monovalentbiologically acceptable anion, and when n is 1, X is a divalentbiologically acceptable anion; each R is independently H or alkyl, orboth Rs together are a piperidino or morpholino residue; and L is aradiosensitizing ligand selected from a mononitro-substituted imidazole,a mononitro-substituted pyrazole, a mononitro-substituted thiazole and amononitro-substituted isothiazole to the animal at a dosage thatradiosensitizes said cells.
 6. The method of claim 5 which furtherincludes irradiating the tumor cells in said animal.
 7. The compositionof claim 1 wherein L is a mononitro-substituted imidazole, amononitro-substituted pyrazole, a mononitro-substituted thiazole or amononitro-substituted isothiazole which is unsubstituted or which issubstituted by one R¹ group, wherein R¹ is selected from alkyloptionally containing an amino substituent, OR³, and N(R³)₂, wherein R³is H or lower alkyl.
 8. The composition of claim 1, wherein L is amononitro-substituted imidazole or a mononitro-substituted pyrazole andwherein one ring nitrogen is unsubstituted or substituted by an R² groupwherein R² is alkyl (1-8C) substituted by one or more --OR³ and whereinone or two methylene groups maybe replaced by O.
 9. The composition ofclaim 8 wherein R² is alkyl of 2-3C mono- or di-substituted with OHand/or OCH₃.
 10. The composition of claim 9 wherein R² is --CH₂CH(OH)CH₂ OCH₃, --CH₂ OCH₂ CHOH, or --CH₂ CH₂ OH.
 11. The composition ofclaim 7 wherein R¹ is amino, alkyl, or aminoalkyl.
 12. The compositionof claim 7 wherein L is selected from misonidazole (MISO), metronidazole(METRO), 4-nitroimadazole (NI), and 2-amino-5-nitrothiazole (ANT). 13.The composition of claim 1 wherein R is H.
 14. The composition of claim12 wherein R is H.
 15. The composition of claim 1 wherein X is Cl. 16.The composition of claim 12 wherein X is Cl.
 17. The composition ofclaim 16 which is selected from mono-MISO, mono-METRO, mono-NI, andmono-ANT.
 18. The composition of claim 15 which is the trans isomer. 19.The method of claim 16 or 19 wherein L is a mononitro-substitutedimidazole, a mononitro-substituted pyrazole, a mononitro-substitutedthiazole or a mononitro-substituted isothiazole which is unsubstitutedor which is substituted by one R¹ group, wherein R¹ is selected fromalkyl optionally containing an amino substituent, OR³, and N(R³)₂,wherein R³ is H or lower alkyl.
 20. The method of claim 16 or 19 whereinL is a mononitro-substituted imidazole or a mononitro-substitutedpyrazole and wherein one ring nitrogen is unsubstituted or substitutedby an R² group wherein R² is alkyl (1-8C) substituted by one or more--OR³ and wherein one or two methylene groups may be replaced by O. 21.The method of claim 20 wherein R² is alkyl of 2-3C mono- ordi-substituted with OH and/or OCH₃.
 22. The method of claim 21 whereinR² is --CH₂ CH(OH)CH₂ OCH₃, --CH₂ OCH₂ CHOH, or --CH₂ CH₂ OH.
 23. Themethod of claim 19 wherein R¹ is amino, alkyl, or aminoalkyl.
 24. Themethod of claim 19 wherein L is selected from misonidazole (MISO),metronidazole (METRO), 4-nitroimadazole (NI), and2-amino-5-nitrothiazole (ANT).
 25. The method of claim 16 or 19 whereinR is H.
 26. The method of claim 38 wherein R is H.
 27. The method ofclaim 16 or 19 wherein X is Cl.
 28. The method of claim 24 wherein X isCl.
 29. The method of claim 28 wherein the compound of formula 1 isselected from mono-MISO, mono-METRO, mono-NI and mono-ANT.
 30. Themethod of claim 2 or 5 wherein the compound of formula 1 is the transisomer.